Various publications, including patents, published applications and scholarly articles, are cited throughout the specification. Each of these publications is incorporated by reference herein, in its entirety. Citations not fully set forth within the specification may be found at the end of the specification.
Coffee aroma and flavor are key components in consumer preference for coffee varieties and brands. Coffee's characteristic aroma and flavor stems from a complex series of chemical reactions involving flavor precursors (Maillard reactions) that occur during the roasting of the bean. Flavor precursors include chemical compounds and biomolecules present in the green coffee bean. To date, over 800 chemicals and biomolecules have been identified as contributing to coffee flavor and aroma (Flament, I. 2002 Coffee Flavor Chemistry J. Wiley, U.K.).
Because coffee consumers are becoming increasingly sophisticated, it is desirable to produce coffee with improved aroma and flavor in order to meet consumer preferences. Both aroma and flavor may be artificially imparted into coffee products through chemical means. See, for example, U.S. Pat. No. 4,072,761 (aroma) and U.S. Pat. No. 3,962,321 (flavor). However, to date, there is little information concerning the influence of natural coffee grain components such as polysaccharides, proteins, pigments, and lipids, on coffee aroma and flavor. One approach is to select varieties from the existing germplasm that have superior flavor characteristics. A disadvantage to this approach is that, frequently, the highest quality varieties also possess significant negative agronomics traits, such as poor yield and low resistance to diseases and environmental stresses. It is also possible to select new varieties from breeding trials in which varieties with different industrial and agronomic traits are crossed and their progeny are screened for both high quality and good agronomic performance. However, this latter approach is very time consuming, with one crossing experiment and selection over three growing seasons taking a minimum of 7-8 years. Thus, an alternative approach to enhancing coffee quality would be to use techniques of molecular biology to enhance those elements responsible for the flavor and aroma that are naturally found in the coffee bean, or to add aroma and flavor-enhancing elements that do not naturally occur in coffee beans. Genetic engineering is particularly suited to achieve these ends. For example, coffee proteins from different coffee species may be swapped. In the alternative, the expression of genes encoding naturally occurring coffee proteins that positively contribute to coffee flavor may be enhanced. Conversely, the expression of genes encoding naturally occurring coffee proteins that negatively contribute to coffee flavor may be suppressed.
Coffees from different varieties and origins exhibit significant flavor and aroma quality variations when the green grain samples are roasted and processed in the same manner. The quality differences are a manifestation of chemical and physical variations within the grain samples that result mainly from differences in growing and processing conditions, and also from differences in the genetic background of both the maternal plant and the grain. At the level of chemical composition, at least part of the flavor quality can be associated with variations in the levels of small metabolites, such as sugars, acids, phenolics, and caffeine found associated with grain from different varieties. It is accepted that there are other less well characterized flavor and flavor-precursor molecules. In addition, it is likely that structural variations within the grain also contribute to differences in coffee quality. One approach to finding new components in the coffee grain linked to coffee quality is to study the genes and proteins differentially expressed during the maturation of grain samples in different varieties that possess different quality characteristics. Similarly, genes and proteins that participate in the biosynthesis of flavor and flavor-precursor molecules may be studied.
Chlorogenic acids are examples of candidate flavor and flavor precursor molecules. Chlorogenic acids (CGA) are an important group of non-volatile compounds found in green coffee grain. CGA are composed of a family of esters between certain trans-cinnamic acids (caffeic and ferulic) and quinic acid (Clifford et al., 2000). In the coffee bean, most CGA can be categorized as belonging to one of three classifications: caffeoylquinic acids (CQA; 3-CQA, 4-CQA, and 5-CQA); dicaffeoylquinic acids (diCQA; 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA); and feruloylquinic acids (FQA). In the mature green coffee grain, the levels of CGA are variable, ranging from approximately 7.88% to 14.4% on a dry matter basis (DMB) for Coffea canephora (robusta), and approximately 3.4% to 4.8% on a DMB for Coffea arabica (Ky et al., 2001). Clifford suggested that the content of CGA in Coffea canephora varies from 7% to 10% on a DMB whereas Coffea arabica varies from 5 to 7.5% on a DMB. (Clifford et al., 1985). In C. canephora, CQA is estimated to comprise 67% of the total CGA content, and diCQA comprise about 20%, and FQA comprise about 13%. In C. arabica, CQA, diCQA and FQA corresponded on average to 80, 15, and 5% of the total CGA content, respectively (Ky et al., 2001).
Much is known about the early phenylpropanoid pathway leading to the synthesis of CGA in plants. (Dixon, R. and Paiva, N. 1995 Plant Cell 7, 1085-1097; Douglas, C. J. 1996 Trends Plant Sci., 1 171-178). An overview of this biosynthetic pathway is summarized graphically in FIG. 1 (Hoffmann et al. 2004). The first step involves the conversion of phenylalanine to cinnamic acid by phenylalanine ammonia lyase (PAL). Four different PAL genes have been characterized in Arabidopsis, and those genes appear to fall into two different groups (Raes, J et al 2003, Plant Physiology 133, 1051-1071). The expression and activities of the different PAL isoforms represent a major branch-point between primary and secondary metabolism in plants, and as such, the different PAL isoforms are under complex regulatory control. (Dixon, R. and Paiva, N. 1995 Plant Cell 7, 1085-1097; Rohde, A., et al. 2004 Plant Cell, 16 p 2749-2771). The next enzyme in the pathway is trans cinnamate-4-hydroxylase (C4H) (accession number BAA24355; CYP73A5 Arabidopsis thaliaina), which converts cinnamic acid to p-Coumaric acid. To date, only one gene has been found for this P450-dependant mono-oxygenase in Arabidopsis, whereas in some other plants, two or more C4H genes have been found that fall into two distinct classes (Reas et al., 2003). The next step in the pathway is carried out by 4-coumarate:Co ligase (4CL). At least four 4CL genes and nine 4CL-like genes have be identified in the Arabidopsis genome (Raes et al. 2003). 4CL forms esters between CoA and phenolic compounds such as p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid acid, and sinapic acid (Hu et al. 1998).
While the enzymes involved with the early part of the phenylpropanoid pathway have been known for several years, the acyl transferases necessary for the next step, hydroxycinnamoyl-CoA transferase (HCT) (tobacco) and hydroxycinnamoyl-CoA quinate: hydroxycinnamoyl transferase (HQT) (from tomato and tobacco) have only recently been purified, and their corresponding DNA sequences cloned and studied (Hoffmann et al 2003 J. Biol Chem 278, 95-103; Niggeweg et al. 2004 Nature Biotechnology, 22, 746-754). These enzymes catalyze ester formation between p-coumaroyl-CoA or caffeoyl-CoA and either shikimate or quinate to generate CGA. HCT has also been shown to catalyze the reverse reaction, i.e., CGA degradation (Hoffmann et al., 2003). Only one gene encoding an enzyme for this step, an HCT, has been identified in Arabidopsis (Raes, et al. 2003). The next step of the phenylpropanoid pathway, the hydroxylation of the 3 position of coumarate, has only recently been elucidated. Schoch et al. found that an Arabidopsis P450 protein (CYP98A3) was capable of hydroxylating the 3 position of coumarate, but only when it was esterified to either shikimate or quinate. (2001 J. Biol Chem 276, 36566-36574). Three genes encoding this enzyme, which is called p-coumarate 3-hydroxylase (C3H), have been identified in the Arabidopsis genome. However, Raes et al. (2003) found that only one of these genes (C3H1) is expressed in all the tissues examined, whereas the other two genes are expressed only in a limited number of tissues and at particular times during development.
The current model of the phenylpropanoid pathway (FIG. 1) suggests that the forward and reverse activities of HCT/HQT play a role in modulating lignin precursor levels, and thus influence the amounts and types of lignin being formed in plants. After the C3H mediated hydroxylation, caffeoyl-CoA can be released from either caffeoyl quinic acid or caffeoyl shikimic acid by HCT/HQT and is then subject to the activity of caffeoyl-CoA 3-0 methytransferase (CCoAOMT) resulting in the formation of feruloyl CoA (Zhang and Chinnappa 1997 J. Biosci. 22, 161-175; Zhong et al. 1998. Plant Cell 10, 2033-2046). CCoAOMT can also methylate 5-hydroxyferuloyl-CoA to sinapoyl-CoA (Zhong et al. 1998). There are seven putative CCoAOMT genes in Arabidopsis, and as in other plants, there appear to be two classes of CCoAOMT genes (Zhong et al. 1998; Raes et al. 2003). Three distinct CCoAOMT classes have now been characterized in tobacco (Maury et al, 1999). Class 1 CCoAOMT genes include the only characterized Arabidopsis CCoAOMT gene, CCoAOMT-1, and the majority of the CCoAOMT genes characterized in other plants. The remaining putative CCoAOMT genes fall into class 2 (Raes et al. 2003). Arabidopsis CCoAOMT-1 is the most highly expressed gene, with expression detected in all tissues examined; lower expression of Arabidopsis CCoAOMT-5 and CCoAOMT-7 can also be detected in all tissues, and low expression of CCoAOMT-2, -3, -4, and -6 can be detected in specific tissues (Raes et al. 2003). Due to its relatively high ubiquitous expression, it is believed that Arabidopsis CCoAOMT-1, and probably orthologs in other plants, play a key role in the lignification that is associated with cell development (Raes et al. 2003).
Initially, many studies of the phenylpropanoid pathway focused on understanding the overall flux of precursors for the synthesis of flavonoids, anthocyanins, the different forms of lignin, and how the pathway was regulated. However, due to the increasing evidence that diets rich in antioxidants can reduce the risk of cancer and degenerative disease by protecting against oxidative stresses (Bazzano, L. et al. 2002; Astley, S., 2003) more recent studies have evaluated intermediary metabolites of the phenylpropanoid pathway, which are present in high levels and have been found to possess high antioxidant activity (Hoffmann et al 2004, Niggeweg et al. 2004). For example, the chlorogenic acids constitute an important group of antioxidants in plant foods such as apples, pears, tomato, potato, and eggplant, as well as green and roasted coffee grain (beans). In addition to the fact that the dietary intake of CGA is best achieved by consumption of plant foods, this class of molecules also show significant bio-availability (Nardini et al. 2002, J. Agric Food Chem. 50, 57355741; Couteau, et al., 2001, J. Applied Microbiol. 90, 873-881; Clifford, M. 2004, Planta Med 70, 1103-1114.). The involvement of CGA in the reduction of oxidative stresses has been demonstrated more directly in plants. For example, Shadle et al. (2003) have demonstrated that overexpression of the enzyme PAL in tobacco, which is responsible for the first step in the biosynthesis of CGA, resulted in an approximately five fold increase in CGA content relative to wild type plants, and demonstrated resistance to the fungal pathogen Cercospora nicotianae. Additional evidence has been generated by Niggeweg et al. (2004), from the overproduction of HQT in the tomato using a constitutive promoter (2×35S promoter). It was demonstrated that higher levels of HQT, an enzyme directly involved in CGA synthesis, resulted in higher levels of CGA, improved resistance to oxidative stress, and increased resistance of the plant to a microbial pathogen. There is a growing realization of the potential for dietary CGA to make important contributions to the antioxidant pool in the plasma, and it is possible that one or more CGA type molecules could be transformed into new or additional metabolites possessing health promoting activities (see, e.g., Clifford 2004). Moreover, it is apparent that CGA may play a role in plant resistance to diseases. Thus, there is a need to better understand, as well as facilitate the synthesis and accumulation of these compounds in plants.
Chlorogenic acids are abundant in coffee. Despite the fact that such molecules play an important role in plant and human health, and in overall coffee flavor, aroma, and quality, few studies have analyzed the phenylpropanoid pathway of coffee, and as such, available data on CGA in coffee is limited. Campa et al. identified a partial putative CCoAOMT sequence (accession number AF534905) (Campa et al., 2003), and mapped this sequence in the coffee genome. In addition, one of the two alleles identified for this gene were found to be associated with the overall level of chlorogenic acids. Bauman et al., have isolated two partial cDNA sequences encoding coffee PAL (accession numbers AAF27655 and AAF27654), and Campa et al, have isolated two cDNA sequences encoding coffee PAL1 (accession number AAN32866) and PAL2 (accession number AAN32867). Sequence alignment of the regions that overlap in the corresponding four proteins was made with Clustal W, and these four sequences were found to be highly related, with 94 to 99.5% predicted identity at the protein level, and greater than 97.1% identity at the nucleic acid level. This level of identity suggests that these four sequences probably represent a single coffee PAL.
Chlorogenic acids are degraded progressively during coffee roasting (De Maria et al., 1995). In fact, these changes represent one of the important compositional changes occurring during this process (Bicchi et al. 1995). It has been reported than 8-10% of the CGA content is lost with every 1% loss of dry matter. (Clifford et al. 1985, 1998). During roasting, CGA are transformed into a series of volatile and non-volatile compounds (S. Homma, 2001). For example, isomerization and hydrolysis can lead to the generation of various quinic acids and quinides, as well as cinnamic acids (Homma, 2001), with the latter group being further degraded via decarboxylation into a range of phenolic compounds, including volatile compounds such as 4-vinylguaiacol, which is associated with a typical coffee aroma (Homma 2001, Farah et al., 2005; and Rizzi, G., et al., 1993 “Flavor chemistry based on the thermally-induced decarboxylation of p-hydroxycinnamic acids. In Food Flavors, Ingredients and Composition (ed. Charalambous) pp. 663-670 Elsevier Science Publishers, Amsterdam, Netherlands).
The degradation products of CGA, particularly the formation of chlorogenic acid lactones and quinic acid lactones, are associated with the perception of bitterness in brewed coffee, and an increase in acidity found in coffee after brewing has been associated with the generation of free quinic acids (Homma, 2001, Leloup et al., 1995; and Buffo et al, 2004). More recently, Muller and Hofmann have found that chlorogenic acids and their thermal degradation products act as thiol binding sites. (2005, J. Agric and Biol Chem 53, 2623-2629). Because thiol-containing compounds, such as 2-furfurylthiol and 3-methyl-2-butene-1-thiol, are important components of a coffee aroma, it has been proposed that the chlorogenic acids and related compounds react with the aroma compounds in a coffee beverage, effectively reducing the aroma of coffee after brewing. (Muller and Hofmann, 2005).
Although it is clear that the chlorogenic acids in coffee influence flavor, it is still unclear if all the chlorogenic acids have an equivalent effect. Therefore, there is a need to generate defined coffee varieties with clear differences in their CGA profiles. There are two different routes to the selection of coffee varieties with different profiles. The first is to use classical breeding and selection, and this approach can be significantly aided by the genetic information about the CGA biosynthetic pathway. Such information will allow alleles of the key genes to be identified and followed in the breeding material and progeny. The second global approach is to directly alter specific key genes involved in the synthesis of CGA. Such may be accomplished via mutagenesis and selection (TILLING), or via the generation of specific gene expression changes using gene cloning and coffee transformation. Grain from these new non-GMO, or GMO varieties, which have altered CGA profiles, can then subsequently be evaluated for their content of chlorogenic acid degradation products and flavor profiles after roasting. Those found to be superior in flavor and other characteristics can then be chosen for further studies.
Increasing chlorogenic acid content in coffee grain could lead to an increase in CGA-derived volatiles implicated in aroma during the roasting process. Thus, modulating CGA content in the coffee grain by genetically modulating the production of the proteins responsible for CGA biosynthesis is a novel means to enhance the aroma and flavor of coffee beverages and coffee products produced from such genetically engineered coffee beans. Enhanced CGA content in the coffee bean may also positively contribute to the overall health and wellness of consumers of coffee beverages and products produced from such coffee beans. In addition, modulating CGA content in the coffee plant has implications for overall health of and disease resistance in the coffee plant. For each of these reasons, there is a need to isolate polynucleotides and polypeptides of the CGA biosynthetic pathway in coffee and to develop methods of utilizing those molecules for one or more of the aforementioned purposes.